title: Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase-peroxidase (KatG).
abstract: The catalase-peroxidase (KatG) of Mycobacterium tuberculosis (Mtb) is important for the virulence of this pathogen and also is responsible for activation of isoniazid (INH), an antibiotic in use for over 50 years in the first line treatment against tuberculosis infection. Overexpressed Mtb KatG contains a heterogeneous population of heme species that present distinct spectroscopic properties and, as described here, functional properties. A six-coordinate (6-c) heme species that accumulates in the resting enzyme after purification is defined as a unique structure containing weakly associated water on the heme distal side. We present the unexpected finding that this form of the enzyme, generally present as a minority species along with five-coordinate (5-c) enzyme, is the favored reactant for ligand binding. The use of resting enzyme samples with different proportional composition of 5-c and 6-c forms, as well as the use of KatG mutants with replacements at residue 315 that have different tendencies to stabilize the 6-c form, allowed demonstration of more rapid cyanide binding and preferred peroxide binding to enzyme containing 6-c heme. Optical-stopped flow and equilibrium titrations of ferric KatG with potassium cyanide reveal complex behavior that depends in part on the amount of 6-c heme in the resting enzymes. Resonance Raman and low-temperature EPR spectroscopy clearly demonstrate favored ligand (cyanide or peroxide) binding to 6-c heme. The 5-c and 6-c enzyme forms are not in equilibrium on the time scale of the experiments. The results provide evidence for the likely participation of specific water molecule(s) in the first phases of the reaction mechanism of catalase-peroxidase enzymes.
authors.affiliation: Department of Chemistry, Brooklyn College, 2900 Bedford Avenue, Brooklyn, New York 11210, USA.
authors.forename: Kalina
authors.initials: K
authors.lastname: Ranguelova
authors.forename: Javier
authors.initials: J
authors.lastname: Suarez
authors.forename: Leonid
authors.initials: L
authors.lastname: Metlitsky
authors.forename: Shengwei
authors.initials: S
authors.lastname: Yu
authors.forename: Shelly Zev
authors.initials: SZ
authors.lastname: Brejt
authors.forename: Sidney Zelig
authors.initials: SZ
authors.lastname: Brejt
authors.forename: Lin
authors.initials: L
authors.lastname: Zhao
authors.forename: Johannes P M
authors.initials: JP
authors.lastname: Schelvis
authors.forename: Richard S
authors.initials: RS
authors.lastname: Magliozzo
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003486:Cyanides / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000378:metabolism*; D007536:Isomerism; D008024:Ligands; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D013059:Spectrum Analysis, Raman; D017186:Titrimetry; D014867:Water / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003486:Cyanides; D005290:Ferric Compounds; D008024:Ligands; D011522:Protons; D014867:Water; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria